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Final Report FRDC Project 2006/064 FINAL REPORT Aquatic Animal Health Subprogram: Development of diagnostic tests to assess the impact of haplosporidian infections in pearl oysters. D. Bearham, S.R Raidal, John Creeper, Frances Stephens, Brian Jones, Brett McCallum, P.K. Nicholls. November 2009 FRDC Project 2006/064 1 FRDC Project 2006/064 Authors: D. Bearham, P.K. Nicholls, S.R Raidal, John Creeper, Frances Stephens, Brian Jones, Brett McCallum. Title: Aquatic Animal Health Subprogram: Development of diagnostic tests to assess the impact of haplosporidian infections in pearl oysters ISBN: 978-0-86905-989-0 © Copyright, Fisheries Research and Development Corporation, Murdoch University, The Western Australian Department of Fisheries, Pearl Producers Association and Douglas Bearham, 2009. This work is copyright. Except as permitted under the Copyright Act 1968 (Cth), no part of this publication may be reproduced by any process, electronic or otherwise, without the specific written permission of the copyright owners. Neither may information be stored electronically in any form whatsoever without such permission. The Fisheries Research and Development Corporation plans, invests in and manages fisheries research and development throughout Australia. It is a federal statutory authority jointly funded by the Australian Government and the fishing industry. The authors do not warrant that the information in this book is free from errors or omissions. The authors do not accept any form of liability, be it contractural, tortious or otherwise, for the contents of this book or for any consequences arising from its use or any reliance placed upon it. The information, opinions and advice contained in this book may not relate to, or be relevant to, reader’s particular circumstances. Opinions expressed by the authors are the individual opinions of those persons and are not necessarily those of the publisher or research provider. 2 FRDC Project 2006/064 Aquatic Animal Health Subprogram: Development of diagnostic tests to assess the impact of Haplosporidian infections in pearl oysters D. Bearham, P.K. Nicholls, S.R Raidal, John Creeper, Frances Stephens, Brian Jones, Brett McCallum. November 2009 FRDC Project 2006/064 August 2008 3 FRDC Project 2006/064 Table of Contents Table of Contents .............................................................................................................. 4 Non-technical summary.................................................................................................... 7 Objectives and extent of progress towards the objectives........................................... 10 Accepted peer-reviewed manuscripts resulting from this research........................... 12 Other Publications and Presentations: ......................................................................... 12 List of Abbreviations, acronyms and definitions ......................................................... 13 Acknowledgments ........................................................................................................... 15 Chapter 1 Background, Needs and Objectives............................................................. 16 1.1 Introduction to bivalve aquaculture. ...........................................................................17 1.2 The pearling industry. .................................................................................................17 1.3 Current disease management in the pearling industry. ...............................................19 1.4 Major pathogens affecting Australian pearl oysters. ..................................................19 1.5 Phylum Haplosporidia (Caullery & Mesnil, 1899).....................................................20 1.5.1 The characteristics of the Haplosporidia. .......................................................20 1.6 The current management of the Haplosporidium sp. in Western Australia 1.7 The requirement for information regarding the infection of Pinctada maxima by a Haplosporidium sp. and the haplosporidian infecting rock oysters Saccostrea cucculata. ...........................................................................................................................................25 Chapter 2 General Site Observations and Sampling Methods................................... 27 2.1 Materials and Methods................................................................................................28 2.1.1 Sampling regime and details of collection sites for the pearl oyster Haplosporidium sp.......................................................................................................................................28 2.1.1.1 Pearl oyster sampling at Cascade Bay .........................................................29 2.1.1.2 Pearl oyster sampling at Willie Creek..........................................................30 2.1.2 Sampling regime and details of collection sites for rock oyster Haplosporidium sp. ...........................................................................................................................................32 2.1.2.1 Rock oyster sampling at the Montebello Islands ..........................................33 2.1.2.2 Rock oyster sampling at Cascade bay, Willie creek and Carnarvon sites...35 2.2 Processing of pearl oyster and rock oyster samples....................................................37 2.2.1 Processing of pearl oyster samples..................................................................37 2.2.1.1 Cascade bay pearl oyster spat. .....................................................................37 2.2.1.2 Willie Creek and Carnarvon pearl oyster samples.......................................38 2.2.2 Processing of rock oyster samples...................................................................38 2.2.2.1 Rock oyster samples from the Montebello islands........................................38 2.2.2.2 Rock oyster samples from Cascade bay, Willie Creek Carnarvon and Koolan Island.........................................................................................................................39 Chapter 3 : Molecular characterisation of a haplosporidian parasite infecting rock oysters Saccostrea cuccullata in north Western Australia. ......................................... 41 3.1 Introduction.........................................................................................................42 3.2 Materials and methods ................................................................................................43 3.2.1 Oyster sampling ...............................................................................................43 3.2.2 DNA extraction ................................................................................................44 3.2.3 DNA extraction: archived samples ..................................................................45 3.2.4 Amplification by Polymerase Chain Reaction .................................................45 3.2.5 DNA sequencing...............................................................................................47 3.2.6 In situ hybridisation .........................................................................................49 3.2.7 Phylogenetic analysis.......................................................................................50 3.3 Results.................................................................................................................51 3.4 Discussion...................................................................................................................59 4 FRDC Project 2006/064 Chapter 4 : The detection of the haplosporidian parasites in rock oysters Saccostrea cuccullata (Born, 1778) using DNA probes................................................................... 61 4.1 Introduction.................................................................................................................62 4.2 Methods and Materials................................................................................................64 4.2.1 DNA extraction. ...............................................................................................64 4.2.2 Amplification by Polymerase Chain Reaction. ................................................65 4.2.3 Generation of polynucleotide probe for ISH. ..................................................66 4.2.4 In-situ hybridisation.........................................................................................67 4.2.5 Minchinia sp. diagnosis ...................................................................................68 4.3 Results.........................................................................................................................69 4.3.1 Polymerase Chain Reaction.............................................................................69 4.3.2 In-situ hybridisation: DNA probe specificity...................................................69 4.3.3 Sensitivity .........................................................................................................75 4.3.4 Diagnosis comparison. ....................................................................................75 4.4 Discussion...................................................................................................................79 4.4.1 Specificity and sensitivity of the PCR assay ....................................................79 4.4.2 The 30 bp oligonucleotide ISH assay...............................................................80
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